High serum cholesterol is commonly associated with an increased risk of heart attack, atherosclerosis and circulatory disorders. In addition, a variety of diseases are caused by cholesterol catabolism disorders, such as gallstone disease, atherosclerosis, hyperlipidemia and some lipid storage diseases.
The major pathway for disposal of cholesterol in the body is by secretion of cholesterol and bile acids into the gut. Bile contains free cholesterol and bile acids. The enzyme, cholesterol 7.alpha.-hydroxylase (CYP7) commits cholesterol to bile acid synthesis and catalyzes the first and rate-limiting step of bile acid synthesis in the liver. Specifically, CYP7 catalyzes, in the presence of reductase and a reducing agent such as NADPH, the initial hydroxylation of cholesterol at the 7.alpha.-position, thereby forming 7.alpha.-hydroxycholesterol. Thus, by increasing synthesis of bile acids, this enzyme plays a key role in the liver by depleting hepatic cholesterol pools, resulting in increased low density lipoprotein (LDL) uptake and a lowering of serum cholesterol levels.
Bile acids are physiological agents which are important in the solubilization of lipid-soluble vitamins, sterol and xenobiotics. Bile acids are synthesized exclusively in the liver and are secreted to the intestines where they are modified to secondary bile acids. Most bile acids are reabsorbed in the ileum and recirculated to the hepatocytes via the portal vein.
The feedback of bile into the liver is known to inhibit cholesterol 7.alpha.-hydroxylase and thus inhibit the overall rate of bile acid synthesis. Cholesterol 7.alpha.-hydroxylase, therefore, has been a subject of active investigations to elucidate the regulatory mechanisms of bile acid synthesis in the liver.
It is known that an interruption of bile acid reabsorption, such as that caused by the bile sequestrant, cholestyramine, or by a bile fistula, stimulates the rate of bile acid synthesis and cholesterol 7.alpha.-hydroxylase activity in the liver. Recent achievements in the purification and cloning of cholesterol 7.alpha.-hydroxylase cDNA have advanced knowledge about the regulation of this enzyme at the molecular level. It has become clear that cholesterol 7.alpha.-hydroxylase activity in the liver is regulated primarily at the gene transcriptional level by bile acids, cholesterol, hormones, diurnal rhythm and other factors. However, the molecular mechanism underlying the regulation of cholesterol 7.alpha.-hydroxylase gene expression remains undetermined.
To understand the structure and function of human CYP7 and its regulation by factors, such as bile acids, cholesterol and hormones, it is essential to purify the human CYP7 enzyme. However, the CYP7 enzyme is present in an extremely low levels in human liver; therefore, it has not been possible to isolate sufficient quantities of purified, functional enzyme from human livers.
Although a cDNA molecule encoding human CYP7 enzyme has been determined, recombinant expression of human CYP7 has not heretofore been achieved. Karam and Chiang, Biochem. Biophys. Res. Commun. 185: 588 (1992). Recently, a strategy to express a catalytically active, truncated rat cholesterol 7.alpha.-hydroxylase in E. coli was disclosed. Li and Chiang, J. Biol. Chem. 266 (29): 19186 (1991). The disclosures of both of those publications are expressly incorporated herein by reference. In the latter publication, it was disclosed that the expression of a membrane-bound hydrophobic protein in E. coli is difficult because the bacteria lacks internal membranes. Via PCR, a modified cDNA was generated that encoded a truncated enzyme lacking the N-terminal 23 amino acid residues of the rat cholesterol 7.alpha.-hydroxylase enzyme. The resulting protein was expressed, predominantly in the cytosol of the bacteria. The purified recombinant enzyme was active, as determined by its ability to hydroxylate cholesterol in a reconstituted system, and has a K.sub.m for cholesterol and V.sub.max similar to those of the rat microsomal (non-truncated) enzyme.
Despite the high sequence identity between the rat and human cholesterol 7.alpha.-hydroxylase, however, it previously has not been possible to express the human cholesterol 7.alpha.-hydroxylase in E. coli following the same strategy and using the same expression vector (pKK233-2) as that previously used for the expression of rat cholesterol 7.alpha.-hydroxylase. Thus, a catalytically active, recombinant human CYP7 enzyme is desirable. Recombinantly-expressed, truncated human CYP7 could be used to detect agents that stimulate or inhibit human CYP7's catalytic activity. Further, such recombinant protein can be used to produce anti-CYP7 antibodies which would be useful for screening assays, for example, to detect stimulated or inhibited production of human CYP7 in response to exposure of a compound to a human CYP7-producing culture.